CN107653575B - A kind of preparation method for the micro-fluidic chip embedding hyaluronic acid functionalized nano-fiber film - Google Patents

A kind of preparation method for the micro-fluidic chip embedding hyaluronic acid functionalized nano-fiber film Download PDF

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CN107653575B
CN107653575B CN201711003398.4A CN201711003398A CN107653575B CN 107653575 B CN107653575 B CN 107653575B CN 201711003398 A CN201711003398 A CN 201711003398A CN 107653575 B CN107653575 B CN 107653575B
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CN107653575A (en
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史向阳
王梦媛
肖云超
朱晓玥
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Donghua University
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Abstract

The present invention relates to a kind of preparation methods of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film, comprising: by chitosan/PEO spinning solution, electrostatic spinning is dried in vacuo, and crosslinking obtains chitosan nano fiber membrane CNFs；Show to modify amphoteric ion CBAA and targeting ligand HA-Cys-MPTMS, obtains the nano fibrous membrane HA-CBAA-CNFs of HA functionalization；It with PDMS microfluidic channel cover plate, is bonded by plasma, obtains the micro-fluidic chip of the nano fibrous membrane of embedding HA functionalization, can be used for circulating tumor cell sorting and lossless release.The present invention has simple process, it is easy to operate, the advantages that high specificity, unmarked, the high-throughput sorting and not damaged release of tumour cell can be completed in a short time, circulating tumor cell is efficiently separated to realize, the early detection of tumor patient is had a good application prospect.

Description

A kind of preparation for the micro-fluidic chip embedding hyaluronic acid functionalized nano-fiber film Method
Technical field
The invention belongs to micro-fluidic chip and cell sorting techniques field, in particular to a kind of embedding hyaluronic acid functionalization The preparation method of the micro-fluidic chip of nano fibrous membrane.
Background technique
Cancer is to lead to the primary factor of human death, according to the World Health Organization (WHO) scholarly forecast, the year two thousand twenty whole world cancer Disease number of the infected is up to 20,000,000 people, and death toll is up to 12,000,000 people, and cancer will become 21 century influence human survival With the chief threat of health.Studies have shown that the death of 90% cancer patient is all closely related with the transfer of tumour.As swollen Key link between tumor primary tumor and transfer stove, circulating tumor cell (Circulating tumor Cells, CTCs) is close Cause extensive concern both domestic and external within several years.At metastases initial stage, tumour cell falls off from primary tumor solid tumor, into blood Liquid becomes CTCs.Although people are very urgent for the demand of the diagnosing and treating of cancer, clinical treatment cancer it is normal Rule means are extremely limited.In addition, once cancer development is to late stage, almost without the possibility of healing.Therefore, the early stage of cancer It was found that, Clinics and Practices be effectively improve survival rate, reduce the death rate main method.
Since the number extremely rareness of CTC in tumor patient blood is (every about 106~107Just there is one in a leucocyte to follow Ring tumour cell), it is difficult to realize separate and capture using conventional means.Such as: CellSearch is the currently the only acquisition U.S. Food and medicine Surveillance Authority approves and is used for the commercially produced product of CTC detection.But testing cost is expensive, is targeted and is tied with magnetic bead It closes CTC and is unfavorable for subsequent detection and analysis.Currently used separation method include physical partition method (based filtration, dielectrophoresis, Hydrodynamics) and biochemistry separating method (immunomagnetic beads, immune microsphere, surface adhesion), and biochemistry separating method is most often Analysis method.
In recent years, the detection platform based on micro-fluidic chip and nano material receives people and widely pays close attention to.Static Spinning Nanofiber operates the features such as being easy and is efficient because its equipment is simple, and has by the nanofiber of its preparation than table Area is big, homogeneity is good, morphology controllable and is concerned the advantages that be easy to functional modification, by electrostatic spinning nano fiber film For the separation and concentration of CTC, cell can be increased and target the making contact probability of material, and then improve the capture rate of cell (Sun N,Liu M,Wang J.N.,et al.Chitosan Nanofibers for Specific Capture and Nondestructive Release of CTCs Assisted by Pcbma Brushes[J].Small:2016.12 (36):5090-5097.).Micro-fluidic chip has many advantages, such as that sample requirements are few, detection sensitivity is high and analysis speed is fast, non- Often it is suitably applied cell sorting (Chen J, Li J, Sun Y.Microfluidic approaches for cancer cell detection,characterization,and separation[J].Lab on a Chip:2012.12(10): 1753-1767.).In addition, the blood sample amount for sorting CTCs is very limited, micro-fluidic chip by chance compensates for this and lacks It falls into, so receiving the favor of numerous researchers with the technology of micro-fluidic chip sorting CTCs.
The method of most of capture CTCs is all based on the specific binding of antibody-antigene at present, however antibody is as one Kind of protein matter, save it is difficult, be easy inactivation and expensive.For antibody, natural polymer ligand and receptor Albumen has stronger binding force, and these natural polymers are cheap, are not easily decomposed, and preservation condition is simple.CD44 by It includes epithelioma, lymph cancer, breast cancer and lung cancer etc. that body, which has excessively high expression in a variety of cancer cell surfaces, with tumour cell Transfer has close relationship.CD44 receptor and hyaluronic acid have very strong binding force, have had many researchs utilizing HA Targeted therapy (Kim Y, the Kumar S.CD44-Mediated of tumour is used for macromolecular surface as targeted molecular modification Adhesion to Hyaluronic Acid Contributes to Mechanosensing and Invasive Motility[J].Mol.Cancer Res.:2014.12(10):1416-1429).Therefore, replace antibody as target using HA Not only specific tumour cell can be captured to molecule, clinical application cost can also be substantially reduced.
Have for the method that the circulating tumor cell of subsequent captured is discharged by light-sensitive material, pH response key and two Sulfide linkage etc..Disulfide bond is favored due to its efficient release efficiency by researcher, makes the common reduction of disulfide bonds at present Agent has glutathione GSH, dithiothreitol (DTT) DTT, sodium borohydride etc..
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of miniflows for embedding hyaluronic acid functionalized nano-fiber film The advantages that preparation method of control chip has simple process, easy to operate, high specificity, moreover it is possible to the circulating tumor cell of capture Lossless release is carried out, is conducive to further do subsequent analysis, higher clinical reference is provided in terms of the early diagnosis of cancer Value.
A kind of preparation method of the micro-fluidic chip of embedding hyaluronic acid functionalized nano-fiber film of the invention, comprising:
(1) chitosan and polyethylene oxide PEO that mass ratio is 9:0.5~1.5 are dissolved in solvent, stirring is anti-at room temperature It answers, is cooled to room temperature, obtain chitosan/PEO spinning solution, electrostatic spinning, vacuum drying obtains chitosan nano fiber membrane, with penta Dialdehyde crosslinking, obtains cross-linked chitosan nano fibrous membrane CNFs；
(2) by hyaluronic acid HA, 1- (3- dimethylamino-propyl) -3- ethyl diimmonium salt hydrochlorate EDCHCl, N- hydroxyl Succinimide NHS is dissolved in solvent, is stirred to react, and the HA-COOH activated is added dropwise to Ethitanin hydrochloric acid Continue to be stirred to react in salt H-Cys-OEt.HCl aqueous solution, is dialysed, is freeze-dried, obtain HA-Cys；Wherein HA, EDC The mass ratio of HCl, NHS, H-Cys-OEt.HCl are 1:2.4~2.5:1.4~1.5:0.8~0.9；
(3) HA-Cys that step (2) obtains is dissolved in solvent, oxydol H is added dropwise2O2, mercaptosilane coupling agents MPTMS, ice-water bath are stirred to react, and are dialysed, are freeze-dried, obtain HA-Cys-MPTMS；Wherein HA-Cys, MPTMS, H2O2's Molar ratio is 1:3:5~8；
(4) CNFs for obtaining step (1) is added in methanol and the mixed solution of NaCl solution, and amphoteric ion carboxylic acid is added Glycine betaine acrylamide CBAA solution, is stirred to react at room temperature, then washing, naturally dry, obtains the shell of amphoteric ion modification Glycan nano fibrous membrane CBAA-CNFs；Wherein the mass ratio of CNFs, CBAA are 4~5:1；
(5) CBAA-CNFs that step (4) obtains is dissolved in solvent, the HA-Cys-MPTMS water that step (3) obtain is added Solution is stirred to react, and then washing, naturally dry, obtain the nano fibrous membrane HA-CBAA-CNFs of HA functionalization；Wherein The mass ratio of CBAA-CNFs, HA-Cys-MPTMS are 1:1.5~1.7；
(6) glass slide for the HA-CBAA-CNFs for obtaining load step (5) is as substrate, with dimethyl silicone polymer PDMS microfluidic channel cover plate, is bonded by plasma, obtains the micro-fluidic chip of the nano fibrous membrane of embedding HA functionalization.
Solvent in the step (1) is the acetic acid that concentration is 85%.
The concentration of chitosan is 2.5~3.5wt% in chitosan/PEO spinning solution in the step (1).
Stirring in the step (1) is magnetic agitation, and the time being stirred to react is 7~9h.
The technological parameter of electrostatic spinning in the step (1) are as follows: spinning voltage 30kV, flow velocity 0.1mL/h, spinning distance 12~15cm, 20~30 DEG C of environment temperature, humidity 20~40%, to be covered with the board device of aluminium-foil paper as reception device.
In the step (1) the vacuum drying time be 12~for 24 hours.
The time being crosslinked in the step (1) is 4~8h.
The molecular weight of HA in the step (2) is 5830.
Solvent in the step (2) is dimethyl sulfoxide DMSO.
Stirring in the step (2) is magnetic agitation；The time being stirred to react is 2.5~3h；Continue to be stirred to react Time is 2~3d.
The process conditions dialysed in the step (2), (3) are as follows: use molecular cut off for the bag filter of 1000Da, in phosphorus After dialysing 1 day in hydrochlorate PBS buffer solution, it is changed to ultrapure water and dialyses 2 days.
Solvent in the step (3) is ethyl alcohol.
The concentration of hydrogen peroxide in the step (3) is 28~32%.
Stirring in the step (3) is magnetic agitation, and the time being stirred to react is 3~5h.
Amphoteric ion CBAA in the step (4) is by the way that dimethylamino propylamine, beta-propiolactone are dissolved in anhydrous third In ketone, polymerization inhibitor 1 is added, 1- diphenyl -2- trinitrophenyl-hydrazine DPPH, ice-water bath reacts 2.5~3.5h, warp under nitrogen protection Purifying, vacuum drying are made；Wherein dimethylamino propylamine, beta-propiolactone, DPPH, anhydrous propanone amount ratio be 1.6g:1~ 1.1g:50mg:18mL.
The process conditions of the purifying are to be purified by flash with anhydrous propanone.
The volume ratio of methanol and NaCl solution is 1:1 in mixed solution in the step (4).
The concentration of NaCl solution in the step (4) is 0.1~0.2M.
The concentration of CBAA solution in the step (4) is 0.07~0.09mg/mL.
The time being stirred to react in the step (4) is 2~3d.
Solvent in the step (5) is isopropanol.
The amount ratio of solvent, HA-Cys-MPTMS aqueous solution in the step (5) is 200mL:800 μ L.
The technological parameter being stirred to react in the step (5) are as follows: whipping temp is 70~80 DEG C, and being stirred to react the time is 7 ~9h.
The micro-fluidic chip for the embedding hyaluronic acid functionalized nano-fiber film that the step (5) obtains is for circulating tumor The sorting of cell CTCs and lossless release.
The sorting of the CTCs and the technological parameter of lossless release are as follows: to the embedding hyaluronic acid functionalized nano-fiber It is passed through the cell suspending liquid containing cancer cell or cancer patient's blood in the micro-fluidic chip of film, utilizes the specific table of cancer cell surfaces Up to the antigen of CD44 and the binding force of HA, the sorting of cancer cell is completed；To the embedding hyaluronic acid functionalized nano-fiber film Micro-fluidic chip in be continually fed into glutathione GSH solution, and collect recovered liquid, complete the lossless release of cancer cell.
Chitosan nano fiber of the invention is low in cost, and a large amount of amino and hydroxyl group abundant, Yi Xiu are contained in surface Decorations modify amphoteric ion CBAA first to reduce the non-specific adsorption of cell, improve the capture purity of cell；Two sulphur are modified again The compound of key discharges convenient for the subsequent tumour cell to capture, finally modifies targeted molecular HA in shell by disulfide bond On glycan nanofiber, according to the easy fracture property of disulfide bond, using reducing substances DTT by the circulating tumor cell of capture from It is released on nano fibrous membrane, is used for targeted capture circulating tumor cell, realize the subsequent analysis to tumour cell.
Beneficial effect
(1) chitosan nano fiber membrane surface of the invention has amino abundant and hydroxyl, is easy to modify, in conjunction with miniflow Chip technology is controlled, has the advantages that device is simple, reproducible.
(2) present invention is using hyaluronic acid decorated nano fibrous membrane specificity capture apparent height expression CD44 receptor Circulating tumor cell, high specificity, operating process is easy, separative efficiency is high.
(3) present invention combines the nano fibrous membrane of functionalization with micro-fluidic chip, for circulating tumor cell tradition The problems such as low efficiency, purity existing for method for separating be low, easy damaged cells, is caught using surface-functionalized nanofiber specificity Circulating tumor cell is obtained, in conjunction with micro-fluidic sorting technology, realizes efficiently separating for circulating tumor cell.
(4) micro-fluidic chip of embedding hyaluronic acid functionalized nano-fiber film produced by the present invention, required blood sample Amount is few, and detection efficiency is high, has a good application prospect.
Detailed description of the invention
Fig. 1 is that the SEM of chitosan nano fiber membrane prepared by the present invention schemes (left side) and diameter distribution profile (right side)；
Fig. 2 is the nuclear magnetic resonance spectroscopy of CBAA prepared by the present invention；
Fig. 3 is infrared spectrogram (b) HA- of the infrared spectrogram (a) of CBAA-CNFs prepared by the present invention, HA-Cys The infrared spectrogram (c) of Cys-MPTMS and the infrared spectrogram (d) of HA-CBAA-CNFs；
Fig. 4 is the anti-protein adsorption rate of CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane prepared by the present invention As a result (a) and anti-leukocyte cell rate result (b)；
Fig. 5 is CNFs (a) prepared by the present invention, CBAA-CNFs (b) and HA-CBAA-CNFs (c) nano fibrous membrane in 0s When water contact angle image；
Fig. 6 (a) is CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane prepared by the present invention to blood compatibility Property evaluation ultraviolet absorpting spectrum；It (b) is enlarged drawing of (a) figure at 500-600nm；
Fig. 7 is CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane prepared by the present invention point in different times Anticoagulation behavior characterization result；
Fig. 8 is CNFs (a) prepared by the present invention, CBAA-CNFs (b) and HA-CBAA-CNFs (c) nano fibrous membrane and cancer To the shows fluorescent microscopy images of cell capture and in different incubation time section (10,20,40 and when cell is incubated for 40min altogether 60min) to the static capture rate (d) of cancer cell；
Fig. 9 (a) is that 40min glutathione discharges the cancer cell of capture and labors to the cancer cell of release under static conditions The shows fluorescent microscopy images extremely dyed；It (b) is the differential responses time interior release efficiency to cancer cell；(c) cancer to release The cell survival rate living extremely dyed；
Figure 10 (a) is trouble of the micro-fluidic chip to simulation of embedding HA-CBAA-CNFs nano fibrous membrane prepared by the present invention Person's blood under different in flow rate (0.5mL/h, 1.0mL/h, 2.0mL/h, 4.0mL/h and 6.0mL/h) Dynamical capture efficiency； (b) for HA-CBAA-CNFs nanofiber micro-fluidic chip prepared by the present invention under 1.0mL/h flow velocity to the cancer of different number The Dynamical capture efficiency of cell；
Figure 11 is stream of the micro-fluidic chip in 1.0mL/h of embedding HA-CBAA-CNFs nano fibrous membrane prepared by the present invention Under speed, to the Dynamical capture separative efficiency of the cancer cell of different number；
Figure 12 is the synthesis schematic diagram of hyaluronic acid functionalized nano-fiber film HA-CBAA-CNFs prepared by the present invention.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Range.
Embodiment 1
(1) 180mg chitosan and 20mg polyethylene oxide PEO are dissolved in 85% acetum, magnetic force stirs at room temperature Reaction 8h is mixed, until chitosan becomes the solution of stable homogeneous, is cooled to room temperature, it is poly- to obtain the shell that concentration is 3.0wt% Sugar/PEO spinning solution is stored in spare in 4 DEG C of refrigerators.Above-mentioned gained spinning solution is slowly drawn in syringe, high pressure is utilized Electrostatic spinning machine carries out electrostatic spinning, spinning condition are as follows: spinning voltage 30kV, flow velocity 0.1mL/h, spinning distance 12cm, ring 20~30 DEG C of border temperature, it is dry to be subsequently placed in vacuum using the board device for being covered with aluminium-foil paper as reception device for humidity 20~40% In dry case for 24 hours, chitosan nano fiber membrane is obtained, is crosslinked 6h in drier with glutaraldehyde, obtain crosslinking shell not soluble in water Glycan nano fibrous membrane CNFs.
(2) 200.94mg hyaluronic acid HA is dissolved in the DMSO of 8mL, by 1- (the 3- dimethylamino third of 492.67mg Base) the N- hydroxysuccinimide NHS of -3- ethyl diimmonium salt hydrochlorate EDCHCl, 296.11mg is dissolved in the DMSO of 1mL, Magnetic agitation reacts 3h, the HA-COOH activated；It is added dropwise to 10mL and contains 176.47mg ethylcysteine hydrochloride In the aqueous solution of H-Cys-OEt.HCl, continues magnetic agitation and react 3d, use molecular cut off for the bag filter of 1000Da, in It after dialysing 1 day in phosphate PBS buffer solution, is changed to ultrapure water and dialyses 2 days, be then freeze-dried, obtain white powder production Object HA-Cys, be stored in -20 DEG C it is spare.
(3) HA-Cys that 500mg step (2) obtains is dissolved in 10mL ethyl alcohol, under magnetic stirring, concentration is added dropwise For 30% oxydol H2O2, 50 μ L mercaptosilane coupling agents MPTMS are then added, ice-water bath magnetic agitation reacts 4h, using retention Molecular weight is the bag filter of 1000Da, after dialysing 1 day in phosphate PBS buffer solution, is changed to ultrapure water and dialyses 2 days, then Freeze-drying, obtain HA-Cys-MPTMS, be stored in -20 DEG C it is spare.
(4) 1.6g dimethylamino propylamine, 1.024g beta-propiolactone are dissolved in 18mL anhydrous propanone, 50mg 1 are added, 1- diphenyl -2- trinitrophenyl-hydrazine DPPH polymerization inhibitor, ice-water bath reacts 3h under nitrogen protection, obtains white precipitate, then uses Anhydrous propanone is purified by flash, and is obtained white powder, is finally dried in vacuo, and amphoteric ion carboxylic acid glycine betaine acrylamide is obtained CBAA。
(5) mixed solution (v/v=of methanol and the NaCl solution of 0.138M is added in the CNFs for obtaining 72mg step (1) In 1:1), the amphoteric ion CBAA solution (concentration 0.08mg/mL) that 16mg step (4) obtain is added, is stirred to react 2 at room temperature Then~3d cleans 2-3 times, naturally dry with ultrapure water, obtain the chitosan nano fiber membrane CBAA- of amphoteric ion modification CNFs。
(6) CBAA-CNFs for obtaining 72mg step (5) immerses in 200mL isopropanol, and 800 μ L steps (3) are added and obtain The aqueous solution containing 118mg HA-Cys-MPTMS, 75 DEG C are stirred to react 8h, then clean 2-3 times with ultrapure water, dry in the air naturally It is dry, obtain the nano fibrous membrane HA-CBAA-CNFs of HA functionalization.
(7) glass slide for the HA-CBAA-CNFs for obtaining load step (6) is as substrate, with dimethyl silicone polymer PDMS microfluidic channel cover plate, is bonded by plasma, obtains the micro-fluidic chip of the nano fibrous membrane of embedding HA functionalization.
Embodiment 2
The present invention with scanning electron microscope (SEM), decaying total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), Nuclear magnetic resonance spectroscopy (1H NMR), uv-visible absorption spectra (UV-vis), anti-protein adsorption test, water contact angle test, blood Liquid phase compatibility test, anticoagulant blood test, static state/Dynamical capture of cancer cell and release test and Immunostaining assay characterization are originally The properties and its combination micro-fluidic chip of the hyaluronic acid functionalized nano-fiber film prepared in invention are thin in circulating tumor Application potential in born of the same parents' sorting and lossless release.
Scanning electron microscope test:
The pattern and diameter of the chitosan nano fiber obtained using SEM characterization 1 step of embodiment (1) are distributed, SEM result As shown in Figure 1, the smooth, uniform diameter by chitosan nano fiber surface that electrostatic spinning technique is prepared, fiber is average straight Diameter is (150.9 ± 29.50) nm.
Nuclear magnetic resonance spectroscopy test:
Using1The synthesis for the amphoteric ion CBAA that H NMR spectra characterization 1 step of embodiment (4) obtains, as a result such as Fig. 2 institute Show: the proton peak at 5.62~6.11ppm of chemical shift represents the proton peak in CBAA in C=C double bond, in chemical shift Proton peak at 2.91ppm represents N- (CH3)2Proton peak, the proton peak at chemical shift 3.40ppm represents N-CH2- CH2The proton peak of-COO, the proton peak at chemical shift 3.2ppm and 1.89ppm represent NH-CH2-CH2-CH2Proton peak. The result shows that successfully preparing amphoteric ion CBAA needed for experiment.
Decay total reflection-Fourier transform infrared spectroscopy test:
Using the structure of CNFs, CBAA-CNFs and HA-CBAA-CNFs in ATR-FTIR characterization embodiment 1, verify transparent Whether the nano fibrous membrane HA-CBAA-CNFs of matter acid functionalization successfully prepares, as a result as shown in Figure 3.
Curve (1) is in 3024cm in Fig. 3 (a)-1Place is the infrared signature absorption peak of-CH in C=C double bond in CBAA, curve (2) in 1590cm-1Place is the characteristic absorption peak that-NH vibrates on amino, and curve (3) is in 1375cm-1There is the feature of C-N in place Absorption peak illustrates that CBAA is successfully modified in nanofiber surface.
Curve (1) is in 1541cm in Fig. 3 (b)-1And 1580cm-1Place is that the infrared signature of amino in H-Cys-OEt.HC is inhaled Peak is received, curve (2) is in 3292cm-1Place is the infrared absorption peak of O-H in HA carboxyl, and curve (3) is in 1739cm-1Locate appearance-CONH Infrared signature absorption peak, show that H-Cys-OEt.HCl is successfully modified on HA.
Curve (1) is in 1739cm in Fig. 3 (c)-1Locate the infrared signature absorption peak of appearance-CONH, curve (2) is in 2562cm-1 Place is the characteristic absorption peak of-SH on MPTMS, and curve (3) is in 564cm-1There is the infrared signature absorption peak of S -- S in place, illustrates to close At HA-Cys-MPTMS compound in be successfully generated S -- S.
Curve (1) is in 2922cm in Fig. 3 (d)-1Place is-CH on HA-Cys-MPTMS2Infrared signature absorption peak, curve (2) in 2943cm-1Place is-CH on CBAA-CNFs2Infrared signature absorption peak, and curve (3) wave number be 2933cm-1With 2878cm-1There is-the CH of enhancing in place2Infrared signature absorption peak, illustrate the nano fibrous membrane HA- of hyaluronic acid functionalization CBAA-CNFs is successfully prepared.
Embodiment 3
Anti- protein adsorption test:
Carrying out anti-albumen to CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiber in embodiment 1, (BSA and fibre are even Albumen) adsorption assessment, for characterize CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane anti-BSA adsorption capacity and Anti- fibronectin adsorption capacity, firstly, with the ultraviolet BSA for measuring various concentration and fine related white calibration curve equation.Herein On the basis of, the concentration for choosing BSA is 2mg/mL and fine related white concentration be 1mg/mL is test concentrations.Take respectively CNFs, Each 10mg of CBAA-CNFs and HA-CBAA-CNFs nanofiber, each four sample of sample are put into 24 orifice plates, then respectively to The BSA and Fibronectin solution of 1mL is added in each orifice plate, is incubated for 1h altogether at room temperature, takes out nanofiber, supernatant is taken to be used in combination 25 type ultraviolet specrophotometer of Lamada tests light absorption value of the supernatant at 278nm, is existed according to different samples Light absorption value at 278nm calculates adsorption rate, and test results are shown in figure 4.
Fig. 4 (a) is anti-protein adsorption rate test result, it is known that compared with the CNFs nano fibrous membrane before modification, CBAA- The adsorption rate of albumen is obviously reduced in CNFs and HA-CBAA-CNFs, shows significant difference, illustrates by amphoteric ion function After energyization modification, nano fibrous membrane obtains excellent anti-albumen adhesion property.Fig. 4 (b) is the test of anti-leukocyte cell rate As a result, knowing that the adsorption rate of albumen is obviously reduced in the CNFs compared with the CNFs nano fibrous membrane before modification, after modification, present Significant difference out further proves there is good anti-protein adsorption by the nano fibrous membrane of amphoteric ion functional modification Performance.
Embodiment 4
Water contact angle test:
The hydrophilicity that chitosan nano fiber modification front and back is had studied by water contact angle measuring instrument, for characterizing The hydrophilicity of CNFs, CBAA-CNFs and HA-CBAA-CNFs nano fibrous membrane.Load is had to the load of thickness even fiber Slide is placed on load sample platform, randomly selects different positions, and 3 μ L drops drop at instrument injection needle is passed through on fibrofelt Survey Software measures the size of contact angle, and shoots drop pattern water contact angle, as a result as shown in Figure 5, it can be seen that CNFs, The contact angle of CBAA-CNFs and HA-CBAA-CNFs nanofiber is respectively 46.9 ± 4.5 °, 35.0 ± 2.4 ° and 24.8 ± 2.4 ° it can be seen that the contact angle of the nanofiber after modification is the nanofiber in the trend being gradually reduced, after showing modification Film has stronger hydrophily, further illustrates that CBAA and HA has successfully been modified on the surface of chitosan nano fiber.
Embodiment 5
Blood compatibility test:
Can blood compatibility be to evaluate a kind of material be applied in capture blood that there are the important indicators of CTCs.By molten The blood compatibility of nano fibrous membrane prepared by blood experimental evaluation.Haemolysis viability experiment for study CNFs, CBAA-CNFs and The biocompatibility of HA-CBAA-CNFs nano fibrous membrane in vivo.
The blood of 1mL Healthy People is taken, is centrifuged 5min (revolving speed 150r/min), supernatant is abandoned, precipitating is washed 5 times with PBS, obtains Red blood cell.It is spare in 4 DEG C of refrigerators with PBS according to the proportional arrangement red blood cell suspension of 1:10.Control group is red thin by 0.2mL Born of the same parents' suspension is dissolved in 0.8mL PBS (negative control) and 0.8mL H respectively2In O (positive control).Then, by CNFs, CBAA-CNFs and HA-CBAA-CNFs chitosan nano fiber is after 4mg/mL is immersed in 10 times of dilution according to mass volume ratio In red blood cell suspension, each nanofiber sample takes 3 Duplicate Samples, is incubated for 2h under the conditions of 37 DEG C.Finally take out fiber Control group and the red blood cell suspension for impregnating fibrofelt are centrifuged 1min (10000r/min), supernatant are taken to be used in combination by felt 25 type ultraviolet specrophotometer of Lamada tests light absorption value of the supernatant at 450-800nm, is existed according to different samples Light absorption value at 540nm calculates hemolysis rate, as a result as shown in fig. 6, (a) is CNFs, CBAA-CNFs and HA-CBAA-CNFs nanometers The ultraviolet absorpting spectrum that fiber evaluates blood compatibility；It (b) is enlarged drawing of (a) figure at 500-600nm.
As shown in Figure 6 within the scope of 450-800nm, the light absorption value of supernatant is significantly higher in control group water, this shows blood Lactoferrin content is higher, i.e., red blood cell rises brokenly completely in water, serious haemolysis occurs.But in CNFs, CBAA-CNFs and In HA-CBAA-CNFs nanofiber and PBS, supernatant light absorption value is very low, illustrates that red blood cell does not occur to rise brokenly.At 540nm Light absorption value calculates it is found that when the nanofiber of functionalization and when Human red blood cells in suspension volume ratio 4mg/mL, the haemolysis of material Rate is 0.98%, 0.82% and 0.64%, and value is all much below critical value 5%.Show CNFs, CBAA-CNFs and HA- CBAA-CNFs nanofiber cannot make human blood cell generate haemolysis, have good blood compatibility.
Embodiment 6
Anticoagulation test:
In order to further verify the blood compatibility of the chitosan nano fiber after modification, using dynamic blood coagulation time method Anticoagulation function evaluation is carried out to the nanofiber of functionalization.Anticoagulation is tested for characterizing CNFs, CBAA-CNFs and HA- The anticoagulation function of CBAA-CNFs nano fibrous membrane.
CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiber are cut into round (φ=14mm) first, are put into 24 holes In plate, each sample takes 4 Duplicate Samples (control group is slide, cover slip).Then to every hole fibrofelt and control group 20 μ L healthy human bloods of upper dropwise addition and 10 μ L CaCl2Solution (0.2mol/L), under the conditions of being placed in 37 DEG C be incubated for 5,10,20,40, 60min.After each incubation time, 3mL distilled water is added to each orifice plate, is then incubated for 5min again, uses ultraviolet spectrometry The light absorption value of photometric determination 540nm, control group slide, CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiber anticoagulation The evaluation result of performance is as shown in Figure 7.
Because the haemocyte that the higher representative of the content of hemoglobin solidifies is fewer, i.e. the anticoagulation function of material is got over It is good.Point in different times as shown in Figure 7, hemoglobin in CBAA-CNFs and HA-CBAA-CNFs nanofiber supernatant Light absorption value is all remarkably higher than slide and CNFs, this illustrates that the nanofiber after targeting modification has preferable anticoagulant active.
Embodiment 7
The capture test of cancer cell static state:
Has the effect of specificity capture cancer cell to verify targeting fibrous material, with highly expressed with CD44 receptor The static capture cancer of functionalized nano-fiber specificity that lung carcinoma cell (A549) is cell model the HA of preparation is examined to modify is thin The effect of born of the same parents.It will be covered with the round glass slide of CNFs, CBAA-CNFs and HA-CBAA-CNFs nanofiberOne Four sample of formula is put into 24 orifice plates to be fixed with steel loop, and the sample of different time points (10,20,40,60min) will be placed on different trainings It supports in plate, radiation sterilization h under the conditions of ultraviolet.The serum free medium that 500 μ L are added in the orifice plate to have sterilized is impregnated fiber 30min.Then culture medium is sucked out, the cell suspending liquid contaminated in advance is added in orifice plate, leucocyte calcein dye is Green, the red dye of A549 cell calcein are red, white blood cell concentration 105A/mL, A549 cell per well are added 300, Suspension dosage is 500 μ L.Be then placed in cell incubator and be incubated for 10,20,40,60min altogether, to regular hour point after respectively Culture plate is taken out, is cleaned three times with PBS, is taken pictures using fluorescence microscope (20 ×) and observe counting, as a result such as Fig. 8 (a)-(c) It is shown, it is known that have more leucocyte and cancer cell on unmodified nanofiber, on the nanofiber for thering is amphoteric ion to modify There are less leucocyte and cancer cell, and have more cancer cell and less leucocyte on targeted nano fiber, absolutely proves The targeted capture cell effect that HA is mediated.Capture rate result such as Fig. 8 (d) institute of three kinds of different materials to A549 cell Show, with the growth of incubation time, three kinds of different materials are in increase trend to the capture rate of A549 cell, in 60min Shi Jiben tends to balance state, but it is much big to the capture rate of cancer cell to target material HA-CBAA-CNFs nano fibrous membrane In the capture rate of non-targeted material C NFs and CBAA-CNFs.When incubation time is 60min, HA-CBAA-CNFs Nanowire Dimension film is up to 80% to the capture rate of A549, and CNFs and CBAA-CNFs nano fibrous membrane is to the capture rate of A549 40% and 18%, the nano fibrous membrane after illustrating modification has good targeting.
Cancer cell static release and dead cell stain test living:
In order to verify disulfide bond different time (10,20,30,40,50,60min) release efficiency and release after cell Activity.Still it is model to examine the releasing effect of targeting material that choosing, which has the highly expressed A549 cell of CD44 receptor,.It will It is covered with the round glass slide of HA-CBAA-CNFs nanofiberOne four parts of examination is put into 24 orifice plates solid with steel loop It is fixed, radiation sterilization 1h under the conditions of ultraviolet.The serum free medium that 500 μ L are added in the orifice plate to have sterilized is impregnated fiber 30min.Then culture medium is sucked out, the A549 cell suspending liquid contaminated with calcein (green) is added in orifice plate, A549 The concentration of cell is 105A/mL, every hole are separately added into the culture solution of 500 μ L.It is then placed in cell incubator and is incubated for 1h.It takes Culture solution is sucked out in orifice plate out respectively, is cleaned three times with PBS, and the glutathione that concentration is 10mM is then added into each orifice plate (GSH), 10,20,30,40,50,60min are reacted respectively, are flushed three times with PBS.(10 ×) are observed under fluorescence microscope, knot Shown in fruit such as Fig. 9 (a), recovered liquid is collected, it is counted with cell counter, shown in the release efficiency of cancer cell such as Fig. 9 (b), In preceding 40min, with the growth of GSH incubation time, the release efficiency of cancer cell is higher, when the time is more than 40min, release effect Rate tends to balance substantially.Cancer cell static release test result shows in summary: 40min is that GSH is broken the best of disulfide bond Time.
It repeats the above steps, but the A549 cell that orifice plate is added does not have to dyeing, the same period, is handled with GSH, collects Recovered liquid dyes the cell released with dead cell kit living, and (10 ×) are observed under fluorescence microscope, with thin Born of the same parents' blood counting chamber calculates cell, shown in release survival rate such as Fig. 9 (c): with the growth (10- of GSH action time 60min), the survival rate of the cancer cell released is lower (99%-80%), in optimal section release time (40min), releases The survival rate of the cancer cell put down is up to 90%, can be used for follow-up cultivation and analysis.
Embodiment 8
The test of cancer cell Dynamical capture:
In order to verify the chitosan nano fiber after HA modification to the capture effect of cancer cell, carried out it is different in flow rate under The measurement of capture rate.Dynamical capture experiment is for studying lower microfluidic system different in flow rate to the capture rate of cancer cell, benefit Result is observed and counted with fluorescence microscope, calculates its capture rate.
There is the chitosan nano fiber membrane of HA as substrate using surface modification, by plasma treatment by substrate of glass, function The chitosan nano fiber membrane of change is bonded with microfluidic channel, obtains micro-fluidic chip, comprising: (1) has 277 cylindroid battle arrays The micro-fluidic chip cell capture channel of column, one section of channel have sample injection port, and the other end has outlet sample, inlet and outlet It is designed in a manner of isoceles triangle；(2) it is modified with the chitosan nano fiber membrane of HA, nano fibrous membrane is clipped in glass slide and poly- two The centre in the channel first polysiloxanes (PDMS) forms interlayer；(3) it is modified with the nano fibrous membrane of HA, is had according to cancer cell surfaces The antigen of particular expression CD44, CD44 receptor and HA have very strong binding force, can the specific cancer for capturing surface expression CD44 Cell, so that cancer cell capture be separated cancer cell on the nanofiber of functionalization.
Model of the A549 cell as cancer cell is chosen, the 20 μ L of calcein (C-AM) of dilution certain multiple (60 times) is taken It is added in A549 cell suspending liquid, is incubated for 12min under the conditions of 37 DEG C.Later plus medium centrifugal 5min (1000r/min), it uses Cell counter takes 1 × 10 to cell count respectively5A A549 cell, under different flow velocitys (0.5mL/h, 1.0mL/h, 2.0mL/h, 4.0mL/h and 6.0mL/h) be passed into microfluidic channel.Since nano fibrous membrane surface modification has a large amount of HA, benefit With the combination of receptor and ligand, when cancer cell flows through in microchannel along fiber surface, cell can be targeted capture, glue It is attached to fiber surface, and non-targeted cell (leucocyte) will flow out channel, to realize the purpose of sorting circulating tumor cell.
The cancer cell of lower capture different in flow rate is counted respectively under fluorescence microscope, finds out the capture effect of cancer cell Rate, as a result as shown in Figure 10 (a), it is known that with the raising of flow velocity, the capture rate of cancer cell is reduced, when flow velocity is 0.5mL/h When, capture rate is up to 93.93%；When flow velocity is 6.0mL/h, capture rate 60.9%.Guaranteeing that capture rate is higher In the case where, 1mL/h is chosen as subsequent experimental flow parameters.Under the conditions of flow velocity is 1.0mL/h, walked by above-mentioned operation Suddenly, HeLa cell, KB cell and MCF-7 cell are chosen respectively by microfluidic channel, respectively to capture under fluorescence microscope Cancer cell count, find out shown in capture rate such as Figure 10 (b) of cancer cell, at identical conditions, targeted nano fiber pair The capture rate highest of Hela cell, capture rate 94.9%；Minimum to the capture rate of KB cell, capture rate is 80.8%.In conclusion cancer cell Dynamical capture test result show HA-CBAA-CNFs nano fibrous membrane can specificity catch Obtain the circulating tumor cell of apparent height expression CD44 receptor.
Embodiment 9
In order to verify the chitosan nano fiber after HA modification to point of the circulating tumor cell in the blood of simulation patient Effect is selected, above-mentioned syringe pump is chosen and is set under the flow velocity of 1.0mL/h, it is strong after the cancer cell of different number to be added to cracking In health human blood, capture rate of the research micro-fluidic chip to cancer cell.
The healthy human blood that 1mL is fresh is taken, is centrifuged 5min (1500r/min), serum is removed, then will dilute certain multiple The DAPI (500 μ L) of (60 times) is added to dialogue cell dyeing 10min in blood, and PBS solution is then added and is centrifuged 5min (1000r/min), to remove extra unbonded dyestuff.Model of the A549 cell as lung carcinoma cell is chosen, takes dilution certain The 20 μ L of calcein (C-AM) of multiple (60 times) is added in A549 cell suspending liquid, is incubated for 12min under the conditions of 37 DEG C, it Afterwards, add culture solution to carry out centrifugation 5min (1000r/min), with cell counter to cell count, take 20,50,100,200 respectively It is added in above-mentioned healthy human blood with 300 A549 cells, microfluidic channel is passed through with the flow velocity of 1.0mL/h, it is aobvious in fluorescence The cancer cell of capture is observed and counted under micro mirror, calculates its capture rate, as a result as shown in figure 11, the microfluidic system Ideal capture rate (85%- is all had to the separative efficiency of different number (20-300/mL) circulating tumor cell 94.9%), capture rate is substantially in the trend of first increases and then decreases, illustrates that the micro fluidic device meets to various concentration cancer cell Capture isolated requirement.
Embodiment 10
Immunostaining test:
Micro flow control chip device in order to further illustrate the present invention has isolated effect to circulating tumor cell in blood Fruit can sub-elect the circulating tumor cell in blood samples of patients, be tested using the blood of patients with lung cancer, and red blood cell will be cracked Blood samples of patients be passed through in micro-fluidic chip system of the invention, then to cell carry out immunostaining, contaminated using immunofluorescence Color identifies the circulating tumor cell of capture.
The cell captured on nanofiber is fixed with 4% paraformaldehyde first, then with 0.1% Triton-100 solution carries out penetrating 10min to cell, then closes 30min with 1% BSA solution, then passes to CK7 (dilution 50 times, 10 μ L) and anti-CD45 (50 times of dilution, 20 μ L) dyeing 1h, last 7min be passed through DAPI (60 times of dilution, 500 μ L), Then in fluorescence microscopy microscopic observation, according to cancer cell judging standard, DAPI dyes all cells, Anti- CD45 only dyes leucocyte, and CK7 can only identify circulating tumor cell.CK+/CD45-/DAPI+ is following in blood Ring tumour cell, CK-/CD45+/DAPI+ are leucocyte, show that the device is able to achieve to circulating tumor cell in blood samples of patients Identification and isolated purpose.

Claims (9)

1. a kind of preparation method for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film, comprising:
(1) chitosan and polyethylene oxide PEO that mass ratio is 9:0.5~1.5 are dissolved in solvent, are stirred to react at room temperature, It is cooled to room temperature, obtains chitosan/PEO spinning solution, electrostatic spinning, vacuum drying obtains chitosan nano fiber membrane, with penta 2 Aldehyde crosslinking, obtains cross-linked chitosan nano fibrous membrane CNFs；
(2) by hyaluronic acid HA, 1- (3- dimethylamino-propyl) -3- ethyl diimmonium salt hydrochlorate EDCHCl, N- hydroxysuccinimidyl Acid imide NHS is dissolved in solvent, is stirred to react, and the HA-COOH activated is added dropwise to ethylcysteine hydrochloride H- Continue to be stirred to react in Cys-OEt.HCl aqueous solution, is dialysed, is freeze-dried, obtain HA-Cys；Wherein HA, EDCHCl, The mass ratio of NHS, H-Cys-OEt.HCl are 1:2.4~2.5:1.4~1.5:0.8~0.9；
(3) HA-Cys that step (2) obtains is dissolved in solvent, oxydol H is added dropwise2O2, mercaptosilane coupling agents MPTMS, Ice-water bath is stirred to react, and is dialysed, is freeze-dried, obtains HA-Cys-MPTMS；Wherein HA-Cys, MPTMS, H2O2Molar ratio For 1:3:5~8；
(4) CNFs for obtaining step (1) is added in methanol and the mixed solution of NaCl solution, and amphoteric ion carboxylic acid beet is added Alkali acrylamide CBAA solution, is stirred to react at room temperature, then washing, naturally dry, obtains the chitosan of amphoteric ion modification Nano fibrous membrane CBAA-CNFs；Wherein the mass ratio of CNFs, CBAA are 4~5:1；
(5) CBAA-CNFs that step (4) obtains is dissolved in solvent, it is water-soluble that the HA-Cys-MPTMS that step (3) obtain is added Liquid is stirred to react, and then washing, naturally dry, obtain the nano fibrous membrane HA-CBAA-CNFs of HA functionalization；Wherein CBAA- The mass ratio of CNFs, HA-Cys-MPTMS are 1:1.5~1.7；
(6) glass slide for the HA-CBAA-CNFs for obtaining load step (5) is micro- with polydimethylsiloxane as substrate Flow control channel cover plate, is bonded by plasma, obtains the micro-fluidic chip of the nano fibrous membrane of embedding HA functionalization.
2. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that: the solvent in the step (1) is the acetic acid that concentration is 85%；Chitosan in chitosan/PEO spinning solution Concentration be 2.5~3.5wt%；Stirring is magnetic agitation, and the time being stirred to react is 7~9h；The technological parameter of electrostatic spinning Are as follows: spinning voltage 30kV, flow velocity 0.1mL/h, spinning 12~15cm of distance, 20~30 DEG C of environment temperature, humidity 20~40%； The vacuum drying time be 12~for 24 hours；The time of crosslinking is 4~8h.
3. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that: the molecular weight of the HA in the step (2) is 5830；Solvent is dimethyl sulfoxide DMSO；Stirring is magnetic force Stirring；The time being stirred to react is 2.5~3h；Continuing the time being stirred to react is 2~3d.
4. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that: the solvent in the step (3) is ethyl alcohol；The concentration of hydrogen peroxide is 28~32%；Stirring is stirred for magnetic force It mixes, the time being stirred to react is 3~5h.
5. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that: the amphoteric ion CBAA in the step (4) is by the way that dimethylamino propylamine, beta-propiolactone to be dissolved in In anhydrous propanone, be added polymerization inhibitor 1,1- diphenyl -2- trinitrophenyl-hydrazine DPPH, under nitrogen protection ice-water bath reaction 2.5~ 3.5h, purified, vacuum drying are made；Wherein dimethylamino propylamine, beta-propiolactone, DPPH, anhydrous propanone amount ratio be 1.6g:1~1.1g:50mg:18mL.
6. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that: the volume ratio of methanol and NaCl solution is 1:1 in the mixed solution in the step (4)；NaCl solution Concentration is 0.1~0.2M；The concentration of CBAA solution is 0.07~0.09mg/mL；The time being stirred to react is 2~3d.
7. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that: the solvent in the step (5) is isopropanol；The technological parameter being stirred to react are as follows: whipping temp be 70~ 80 DEG C, being stirred to react the time is 7~9h.
8. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 1 Method, it is characterised in that: the micro-fluidic chip for the embedding hyaluronic acid functionalized nano-fiber film that the step (5) obtains is for following The sorting and lossless release of ring tumour cell CTCs.
9. a kind of preparation side of micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film according to claim 8 Method, it is characterised in that: the sorting of the CTCs and the technological parameter of lossless release are as follows: received to the embedding hyaluronic acid functionalization It is passed through the cell suspending liquid containing cancer cell or cancer patient's blood in the micro-fluidic chip of rice tunica fibrosa, utilizes cancer cell surfaces The antigen of particular expression CD44 and the binding force of HA, complete the sorting of cancer cell；To the embedding hyaluronic acid functionalized nano It is continually fed into glutathione GSH solution in the micro-fluidic chip of tunica fibrosa, and collects recovered liquid, completes the lossless of cancer cell and releases It puts.
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